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Serine integrase chimeras with activity in E. coli and HeLa cells.

Farruggio AP, Calos MP - Biol Open (2014)

Bottom Line: The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms.Our work is the first to demonstrate chimeric serine integrase activity.This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5120, USA.

No MeSH data available.


Related in: MedlinePlus

HeLa pseudo site integration assay.(A–C) Maps of pDB2, pDCP and pDCbC-P3 donor plasmids. (D) Mean counts of G418-resistant colonies. HeLa cells were transfected with different combinations of an integrase expression vector and a donor plasmid, and were then subjected to G418 selection. The abbreviations “−”, “C31” and “BC” refer to the negative control, phiC31 and BC{−1} integrase expression plasmids, respectively. The pDB2, pDCP and pDCbC-P3 vectors carry a 285 bp phiC31 attB, 50 bp phiC31 attP and 50 bp CbC P3 site, respectively. In all trials, each plasmid combination was transfected in triplicate. The error bars indicate standard error of the mean. Data from one of two independent representative trials is shown here.
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f06: HeLa pseudo site integration assay.(A–C) Maps of pDB2, pDCP and pDCbC-P3 donor plasmids. (D) Mean counts of G418-resistant colonies. HeLa cells were transfected with different combinations of an integrase expression vector and a donor plasmid, and were then subjected to G418 selection. The abbreviations “−”, “C31” and “BC” refer to the negative control, phiC31 and BC{−1} integrase expression plasmids, respectively. The pDB2, pDCP and pDCbC-P3 vectors carry a 285 bp phiC31 attB, 50 bp phiC31 attP and 50 bp CbC P3 site, respectively. In all trials, each plasmid combination was transfected in triplicate. The error bars indicate standard error of the mean. Data from one of two independent representative trials is shown here.

Mentions: Because the phiC31 and phiBT1 integrases are capable of pseudo att-site integration in mammalian cells (Chalberg et al., 2006) (phiBT1 int: unpublished observations), we next tested whether BC{−1} integrase could perform this function in HeLa cells. To detect pseudo site integration, we co-transfected different combinations of G418-resistance donor plasmids, which carry an att-site (Fig. 6A–C), with integrase expression vectors, and then counted the resistant colonies present after two weeks of G418 selection. A low donor:integrase plasmid ratio was used to minimize the contribution of random integrants to our colony counts.


Serine integrase chimeras with activity in E. coli and HeLa cells.

Farruggio AP, Calos MP - Biol Open (2014)

HeLa pseudo site integration assay.(A–C) Maps of pDB2, pDCP and pDCbC-P3 donor plasmids. (D) Mean counts of G418-resistant colonies. HeLa cells were transfected with different combinations of an integrase expression vector and a donor plasmid, and were then subjected to G418 selection. The abbreviations “−”, “C31” and “BC” refer to the negative control, phiC31 and BC{−1} integrase expression plasmids, respectively. The pDB2, pDCP and pDCbC-P3 vectors carry a 285 bp phiC31 attB, 50 bp phiC31 attP and 50 bp CbC P3 site, respectively. In all trials, each plasmid combination was transfected in triplicate. The error bars indicate standard error of the mean. Data from one of two independent representative trials is shown here.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4197438&req=5

f06: HeLa pseudo site integration assay.(A–C) Maps of pDB2, pDCP and pDCbC-P3 donor plasmids. (D) Mean counts of G418-resistant colonies. HeLa cells were transfected with different combinations of an integrase expression vector and a donor plasmid, and were then subjected to G418 selection. The abbreviations “−”, “C31” and “BC” refer to the negative control, phiC31 and BC{−1} integrase expression plasmids, respectively. The pDB2, pDCP and pDCbC-P3 vectors carry a 285 bp phiC31 attB, 50 bp phiC31 attP and 50 bp CbC P3 site, respectively. In all trials, each plasmid combination was transfected in triplicate. The error bars indicate standard error of the mean. Data from one of two independent representative trials is shown here.
Mentions: Because the phiC31 and phiBT1 integrases are capable of pseudo att-site integration in mammalian cells (Chalberg et al., 2006) (phiBT1 int: unpublished observations), we next tested whether BC{−1} integrase could perform this function in HeLa cells. To detect pseudo site integration, we co-transfected different combinations of G418-resistance donor plasmids, which carry an att-site (Fig. 6A–C), with integrase expression vectors, and then counted the resistant colonies present after two weeks of G418 selection. A low donor:integrase plasmid ratio was used to minimize the contribution of random integrants to our colony counts.

Bottom Line: The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms.Our work is the first to demonstrate chimeric serine integrase activity.This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5120, USA.

No MeSH data available.


Related in: MedlinePlus